Protein and peptide quantification is an important process required by thousands of laboratories, research and development departments, and industries for activities ranging from protein characterization to clinical diagnostic testing to drug dosing. There are several methods for evaluating protein and peptide content quantitatively and qualitatively and multiple factors to consider for each type of application, including the accuracy required, concentration of protein in the sample, assay specificity, presence of interfering chemicals in the solvents used and the ease and reliability of the assay method. Gel electrophoresis has been used for size-based separations of protein mixtures for over forty years, and is still the most frequently used technique for protein separations in many biological research laboratories. However, quantification of proteins separated by the gel method still has many challenges.
The fast-paced development of protein and peptide applications in therapeutic and non-therapeutic industries, in addition to great progress in proteomics methods, has increased the importance of developing an accurate methodology for quantifying proteins. There are several methods to measure individual proteins, either in solution or using a solid-phase assay such as a gel. However, each method has certain limitations.
Amino acid analysis is the most accurate available method for protein quantification. The procedure consists of several steps: hydrolysis, derivatization, separation, and detection followed by quantification. It is based on the measurement of individual amino acid content in the protein/peptide structure and the calculation of total protein using these individual measurements. However, this is a very expensive method that also requires long processing times, and the results are also greatly affected by the level of the operator's technical expertise. Detected protein content highly depends on the response of any given sequence to hydrolysis, derivatization conditions, or sample contaminants (such as the presence of nonvolatile amines like Tris or glycine). Therefore, there are few core facilities that perform amino acid analysis and usually laboratories do not run their own amino acid analysis equipment. In addition, this method must be performed on pure proteins, which would require extraction of proteins from the electrophoresis gels. Further, this is a destructive method of analysis.
The UV-visible absorbance method is usually used to determine the protein content in solutions containing a single type of protein or to calculate the total protein content of the solution. This is a nondestructive method, allowing the proteins to be recovered for further analysis. Aromatic amino acid residues (tyrosine and tryptophan) and peptide bonds absorb UV light and the absorbance at 280 nm is measured as an indicator of protein content. In this method, any protein solution can be analyzed and the precision is about 10-100 mg of protein. The UV spectrometer is inexpensive and easy to use, and can also be coupled with colorimetric methods to enhance accuracy. Colorimetric methods are based on the chemical binding of a dye to the protein sequence. There are different types of dye attachment, including protein-copper chelation (Bicinchoninic assay (BCA) and Lowry assay) and dye-binding based detection (Bradford and 660 Assay). The Bradford assay measures the degree of binding to Coomassie Brilliant Blue dye, which changes color from brown to blue in the presence of proteins. However, the UV absorbance method cannot be used on proteins separated by gel electrophoresis because gel material absorbs UV light over the same range of wavelengths. Additionally, proteins that do not contain aromatic amino acids cannot be quantified based on UV absorbance. Colorimetric assays are easy to use, but they are highly sensitive to sample components (such as detergents and reducing agents), protein composition, protein structure, and dye-binding properties. Therefore, the assays are semi-quantitative, and not as precise as gold standard methods such as amino acid analysis since protein absorbance at 280 nm depends on protein amino acid composition and secondary and tertiary protein structures. In addition, the assay outcome depends on the number of basic amino acid residues in the analyzed protein, which can vary greatly among proteins, and make interpretations of results challenging.
The mass spectrometry method uses excitation of protein/peptide ions by different sources, such as electron spray, and measures signal intensities across samples in a mass to charge (m/z) range. Because sample processing, separation, and transfer to the mass spectrometer are generally automated, quantitative data can only be obtained from liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry experiments by determination of the abundance of different proteins from their mass spectra. Mass spectrometry is frequently used for functional proteomics, which seeks to measure small changes in protein abundance in a complex biological system in response to perturbations such as disease progression or drug treatment. Notwithstanding the sensitivity of the method, mass spectrometry is very expensive and slow, and expertise is required to run the mass spectrometer and interpret the results. Additionally, mass spectrometers may not be entirely quantitative. Mass spectrometry analysis also requires purified proteins separated by gels to be extracted, and it is a destructive technique.
Another method for protein quantification is based on image analysis of proteins directly on electrophoresis gels, which can be performed on a microscopic image of the gel to quantify the protein content. The method depends on the assumption that the protein bands are well resolved, requires the addition of an external contrast agent, and conversion of the microscopic images to digital data for analysis increases the processing error. Visual protein detection for gel electrophoresis ranges from 1 ng to 5 ng/band for silver stained gels and 40 ng to 50 ng/band for Coomassie Blue stained gels. This requires a very accurate calibration curve for the stain intensity to accompany the microscopic image of the gel, which can be user dependent for solutions with low protein content and includes low image resolution at the gel band edges, and thus further external contrast must be added to the protein.
Immunological based methods employ an antibody specific for a protein that are fixed on a polymeric substrate, and interaction of the protein sample with the antibody is analyzed. Usually the target protein is detected with a second antibody that recognizes a different epitope to the capture antibody. However, even if antibodies could be found to bind to every protein in the sample, the signal intensity for each antigen-antibody interaction would depend not only on the abundance of the target protein but also on the strength of the antigen-antibody binding, and it would be very difficult to quantify. This method also cannot be applied to protein bands on gel materials.
Recently, EMD Millipore has introduced The Direct Detect™ spectrometer that uses infrared-based methods in the mid-infrared spectral range for protein quantification in a solution. The process is very similar to the UV absorbance methods described herein, where a drop of protein solution is used to measure the total protein (or individual protein if the solution contains a single type of protein). However, this technology requires the use of a membrane comprising hydrophilic polytetrafluoroethylene (PTFE) that is transparent in the mid-infrared region, and is only applicable to measure total protein content of the solution, not individual proteins mixed in a solution. The Amide I infrared spectral region is used for quantification, but this spectral region is also sensitive to water content in the sample.
Overall, there is a need in the art for simpler, improved methods for more precise quantification of proteins and peptides that have been separated by gel electrophoresis, the method utilized most frequently in biological labs. The present invention meets this need.